Poly-Gamma-Glutamic Acid Secretion Protects Bacillus subtilis from Zinc and Copper Intoxication

ABSTRACT Zinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms. Here, we show that the Gram-positive soil bacterium, Bacillus subtilis, produces the extracellular polymeric substance, poly-gamma-glutamate (γ-PGA) as a protective mechanism in response to zinc and copper excess. Furthermore, we provide evidence that zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two-component regulatory system and likely occurs at a posttranscriptional level through the small protein, PgsE. These data provide new insight into bacterial metal resistance mechanisms and contribute to our understanding of the regulation of bacterial γ-PGA biosynthesis. IMPORTANCE Zinc and copper are potent antimicrobial compounds. As such, bacteria have evolved a diverse range of tools to prevent metal intoxication. Here, we show that the Gram-positive model organism, Bacillus subtilis, produces poly-gamma-glutamic acid (γ-PGA) as a protective mechanism against zinc and copper intoxication and that zinc and copper dependent γ-PGA production occurs by a yet undefined mechanism independent of known γ-PGA regulation pathways.

In this manuscript, titled 'Poly-gamma-glutamic acid secretion protects Bacillus subtilis from zinc and copper intoxication', Reina Deol and et al. report that Bacillus subtilis produces γ-PGA as a protective mechanism in response to zinc and copper excess. Towards molecular mechanism elucidation, the authors provide evidence that the observed zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two component system and claimed that this likely occurs at a post-transcriptional level. Taken together, the paper is well written and easy to follow, and I think this report can broaden the understanding of bacterial metal resistance. Publication can be considered after some revisions are properly addressed.
Main comments: 1. The excitement is diminished after reading the first sentence (line 157) in Discussion (need reference here by the way, ref 34?). If γ-PGA production as a response to zinc excess has been reported before, then the main conclusions of this paper should be tailored, should only focus on copper and then zinc was, in fact, used as a pos control only. 2. To measure γ-PGA production in liquid media, why chose only 250 μM ZnSO4 or 100 μM CuSO4? I would expect a concentration titration effect, and a dose-dependent response can provide more information. 3. At the end, molecular mechanism characterization is certainly incomplete, any additional investigation results towards elucidating the mechanism should be added to further strengthen the story.

Reviewer #2 (Comments for the Author):
This is a nice study that nevertheless leaves room for improvement. First question: Why did the authors use LB not minimal salt medium. If the author's aim is to dissect the answer to a single metal minimal salts medium might have been appropriate. How widespread is gamma-PGA in Gram-positive bacteria. This pathway only appears to be present in Bacillus. True? The growth curves are nice. This must have been one of the few times where the impact of components of EPS on metal resistance has actually been measured. Is this correct?
The induction experiments are indeed surprising given that there is a clear phenotype. Just out of curiosity, Is this also occurring in minimal salts medium.
Minor points: species in references should be in italic Staff Comments:

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Here, we will address each reviewers comments individually (response in bold).
Reviewer 1: 1. The excitement is diminished after reading the first sentence (line 157) in Discussion (need reference here by the way, ref 34?). If γ-PGA production as a response to zinc excess has been reported before, then the main conclusions of this paper should be tailored, should only focus on copper and then zinc was, in fact, used as a pos control only.
Absolutely! We have modified the text throughout the manuscript to highlight that the zinc effects have been observed previously and that our contribution is to extend this to include copper. We also highlight that our paper is the first to show experimentally that PGA provided protection from zinc and copper intoxication.
2. To measure γ-PGA production in liquid media, why chose only 250 µM ZnSO4 or 100 µM CuSO4? I would expect a concentration titration effect, and a dose-dependent response can provide more information.

Thank you for your comment. Those values were chosen to be consistent with previous studies on the B. subtilis response to zinc and metal intoxication. We have noted this in the text and the appropriate citations have been added (see lines 108-111).
3. At the end, molecular mechanism characterization is certainly incomplete, any additional investigation results towards elucidating the mechanism should be added to further strengthen the story.
We certainly agree that we have not fully characterized the molecular mechanism. In our revision, we include new data to suggest that the small protein PgsE, which was previously implicated in zinc dependent PGA production, is also involved in copper dependent PGA production.
Reviewer 2: This is a nice study that nevertheless leaves room for improvement.

Thank you for the kind words. We have made every effort to incorporate your suggestions.
First question: Why did the authors use LB not minimal salt medium. If the author's aim is to dissect the answer to a single metal minimal salts medium might have been appropriate.

We have repeated the experiments using minimal salt medium and see a similar metal dependent PGA induction.
How widespread is gamma-PGA in Gram-positive bacteria. This pathway only appears to be present in Bacillus. True? Yes, this is true. PGA production does seem to be limited to the Bacilli. There are some reports that S. pneumoniae may also produce PGA, but information is limited.
The growth curves are nice. This must have been one of the few times where the impact of components of EPS on metal resistance has actually been measured. Is this correct?
Yes, this is correct. While other studies have suggested this due to the ability of PGA to bind metals, our study is the first to demonstrate that PGA serves as a bacterial mechanism to prevent metal intoxication.
The induction experiments are indeed surprising given that there is a clear phenotype. Just out of curiosity, Is this also occurring in minimal salts medium.
We have repeated the experiments in a minimal medium and see a similar metal dependent induction of PGA production. Future studies will use this medium as a basis for sorting out the molecular details underlying the phenotype.

Minor points: species in references should be in italic
Thank you for reading the manuscript so carefully! Re: Spectrum01329-21R1 (Poly-γ-glutamic acid secretion protects Bacillus subtilis from zinc and copper intoxication) Dear Prof. Pete Chandrangsu: I am pleased to inform you that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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